Method for preparing hepatitis B immune globulin

ABSTRACT

Hepatitis B immune globulin is prepared from individuals who exhibit an increase in hepatitis B surface antibody of at least 2,000 PHA units/ml following immunization with hepatitis B surface antigen.

RELATED APPLICATION

This application is a division of application Ser. No. 830,291 filedSept. 2, 1977, now U.S. Pat. No. 4,118,477, which in turn is acontinuation-in-part of application Ser. No. 768,236 filed Feb. 14,1977, now abandoned, which in turn is a division of application Ser. No.587,707 filed June 16, 1975, now U.S. Pat. No. 4,024,243.

BACKGROUND OF THE INVENTION

This invention relates to hepatitis B and, more particularly, to avaccine for hepatitis B and to a method for purifying hepatitis Bantigen for use as a vaccine.

Hepatitis B is one of the types of viral hepatitis which results in asystemic infection with the principal pathologic changes occurring inthe liver. This disease affects mainly adults and is maintained chieflyby transfer of infection from long term carriers of the virus. Usualmethods of spread are by blood transfusion, contaminated needles andsyringes, through skin breached by cuts or scratches, by unsterilizeddental instruments as well as by saliva, venereal contact or exposure toaerosolized infected blood.

The incubation period of type B hepatitis is relatively long: from 6weeks to 6 months may elapse between infection and the onset of clinicalsymptoms. The illness usually begins with fatigue and anorexia,sometimes accompanied by myalgia and abdominal discomfort. Laterjaundice, dark urine, light stools and tender hepatomegaly may appear.In some cases, the onset may be rapid, with appearance of jaundice earlyin association with fever, chills and leukocytosis. In other casesjaundice may never be recognized and the patient may be aware only of a"flu-like" illness. It is estimated that the majority of hepatitisinfections result in a mild, anicteric illness.

It is, accordingly, an object of the present invention to provide animproved method for extracting hepatitis B surface antigen. Anotherobject is to provide a faster and more economical method of extractingantigen. A further object is to provide antigen which is free of addedcesium or potassium. These and other objects of the present inventionwill be apparent from the following description.

SUMMARY OF THE INVENTION

Human biological fluid containing hepatitis B surface antigen issubjected to an isopycnic banding wherein the gradient is NaBr. Fasterand more economical processing of the plasma is obtained by multipleloading of the step gradient whether the step gradient be NaBr or aconventional gradient.

DETAILED DESCRIPTION

The starting material for the purified hepatitis B surface antigen(HB_(s) Ag) of the present invention is human biological fluidcontaining hepatitis B surface antigen. The fluid may be any humanbiological fluid containing HB_(s) Ag such as, for example, plasma,saliva, fecal extracts, nasal pharyngeal secretions, bile, spinal fluid,sweat, urine, semen, vaginal secretions or menstrual blood. The mostreadily obtainable biological fluid is plasma. The plasma is obtained inconventional manner, e.g., by plasmaphoresis. The level of HB_(s) Ag inthe human biological fluid may be measured in known manner by anysuitable means, e.g., reversed passive hemagglutination or complementfixation. When the biological fluid is plasma, it optionally may becooled and the cryoprecipitate which forms may be removed by lightcentriguation. The HB_(s) Ag is the human biological fluid is isolatedby an isopycnic banding step followed by a rate zonal banding step.

In isopycnic banding the partially purified concentrate is contactedwith a liquid medium having a density gradient therein which includesthe density of the specific antigen being isolated. The liquid medium isthen subjected to ultracentrifugation to attain an equilibriumdistribution of the serum components through the density gradient,according to their individual densities. Successive fractions of themedium are displaced and those containing the desired antigen, i.e. thefractions having a density of from about 1.21 to about 1.24 g/cc, areseparated. The application of this technique to the purification ofHB_(s) Ag is described in German Specification No. 2,049,515 and U.S.Pat. No. 3,636,191. The concentrations of the solutions forming thegradient are selected so as to encompass the density range of from about1.0 to about 1.41 g/cc. The liquid medium may be employed in the form ofa linear gradient or a step gradient. Preferably it is employed in theform of a step gradient due to its inherent higher capacity forfractionation.

In rate zonal banding the partially purified concentrate is subjected toultracentrifugation in contact with a liquid medium having a densitygradient therein, but this time using the rate zonal technique, i.e., ata rate and for a period such that equilibrium is not attained, theHB_(s) Ag and other residual serium components being distributed throughthe medium according to their sedimentation coefficients in the medium.The concentrations of the solutions forming the step gradient areselected so as to encompass the density range of from about 1.0 to about1.28 g/cc. The rate zonal step is carried out until the HB_(s) Agresides in the 1.13 to 1.16 density region. At this point the HB_(s) Agis separated from the bulk of the crude plasma proteins and, mostsignificantly, is also separated from the macroglobulin complement ofthe plasma. If the rate zonal step is carried out such that the desiredHB_(s) Ag antigen reaches its equilibrium position, i.e., about 1.18 toabout 1.20 g/cc, it has been found that a plasma macroglobulin fractionwill appear as a contaminant in the desired HB_(s) Ag antigen fraction.

The liquid media used in the isopycnic banding and rate zonal steps maybe any density gradient in the appropriate ranges. Prior art solutes forsuch solutions include, e.g. sucrose, potassium bromide, cesiumchloride, potassium tartrate and the like.

The isopycnic banding step is conveniently carried out in a centrifuge,for example, Electronucleonics-K, by filling the stationary rotor withsaline solution, then successively displacing the saline solutionupwards with aliquots of a liquid medium solution of increasing densityuntil a step gradient is formed. The biological fluid is introduced atthe top of the rotor displacing some of the highest density solutionfrom the bottom. Typically, the volume of fluid is from about 15% toabout 40% that of the step gradient. The centrifuge is brought up tospeed through a programmed speed control system which prevents mixingduring the initial reorientation phase. When equilibrium is attained andthe product is in its proper density position, the rotor is slowed downthrough the same system to prevent mixing upon reorientation to theoriginal configuration. Then the gradient is drained from below and theproper density cut collected. A similar technique is used in the ratezonal banding. The proper density cut from the rate zonal banding isdesired concentrate of hepatitis B antigen.

Due to the small size, appoximately 20 nm of HB_(s) Ag the isopycnicbanding step is quite time consuming, requiring about 18 hours ofcentrifuging. As a result, even operating 24 hours a day, 7 days a week,it is possible to prepare only about 4 batches of biological fluid percentrifuge per week. Productivity can be increased, of course, byutilizing additional centrifuges. This involves a tremendous capitalinvestment, however, as each centrifuge is extremely expensive.

It has now been found that substantial increases in productivity andsubstantially reduced operating costs are obtained by multiple loadingof the isopycnic banding gradient. Multiple loading means subjecting asample of biological fluid containing HB_(s) Ag to isopycnic bandingconditions for a time sufficient to permit substantially all of the HB₂Ag in the fluid to pass into the gradient but insufficient to achieveequilibrium, and repeating this step at least once with an additionalsample of fluid containing HB_(s) Ag, before continuing the isopycnicbanding conditions for a time sufficient to achieve equilibrium. Ifdesired, a gradient may be loaded with up to about 6 samples of fluid.As the time required for the HB_(s) Ag in the fluid to enter thegradient is only a fraction of that required to reach equilibrium, andas the subsequent time required to reach equilibrium is the same whetherthe gradient is singly or multiply loaded, substantial savings in timeand reductions in unit processing costs are obtained.

While the increased productivity and reduced costs of the multiplebanding technique of the present invention may be achieved with anysuitable gradient, preferably the gradient is sodium bromide.

The isopycnic banding is carried out to equlibrium by centrifuging atfrom about 40,000×g to about 80,000×g for about 10 hours or beyond. Ithas been found, however, that by centrifuging the fluid for about 4hours substantially all of the HB_(s) Ag is caused to move into theisopycnic banding gradient. Then the sample of spent fluid is removedand a fresh sample of fluid equal in volume to the first sample islayered onto the gradient. Centrifuging may then be continued aspreviously for about 10 hours or beyond to cause the HB_(s) Ag in bothsamples to move into the equilibrium density region of the gradient(1.21 to about 1.24 g/cc) to complete the banding. Alternatively thecentrifuging may be continued for 4 hours, the spent fluid removed and athird sample of fresh fluid layered onto the gradient. This multipleloading procedure may be repeated six or even more times beforecompleting the banding by centrifuging for about 18 hours.

The ratio of the charge (fluid) volume to the gradient volume is fromabout 1:3 to about 1:6. When a single fluid charge is applied to thegradient and centrifuged under isopycnic banding conditions (e.g. forfrom about 16 to about 20 hours at about 30,000 rpm in the K-IIcentrifuge) the resulting product generally will have a protein contentdepending on the amount of protein in the original fluid. In the case ofplasma the protein content is approximately 4-10 mg/ml in a volume of1.0 liter.

When a double charge of fluid is applied to the gradient and centrifugedunder isopycnic banding conditions, (for from about 16 to about 20 hoursat 30,000 rpms) the resulting product will have a protein content whichis additive for the charges employed depending on the amount of proteinin the original plasma. The level of protein increases in this mannerfor each subsequent charge of fluid applied to the gradient. In the caseof plasma the protein content is typically from about 8-20 mg/ml in avolume of 1.0 liter.

The product is then subjected to a rate zonal banding. The rate zonalbanding is carried out until the HB_(s) Ag is in the density range offrom about 1.13 to about 1.16 g/cc. Typically this takes for from about16 hours to about 20 hours, preferably for from about 17 to about 18hours, at from about 30,000×g to about 60,000×g.

According to one aspect of the present invention the gradient is formedof sodium bromide whether or not the multiple loading technique is used.In contrast to heretofore used materials sodium bromide has definiteadvantages. The solubility of sodium bromide allows the use of highdensity solutions in the formation of gradients at refrigeratortemperatures (2°-6° C.). There are definite economic advantages to usingsodium bromide over a salt such as cesium chloride as well as not havingto contend with the problem of human toxicity from residual and HB_(s)Ag bound cesium ions. In sodium bromide gradient any ions bound to theHB_(s) Ag due to biophysical characteristics, will be a sodium saltwhich is very compatible with the human biological system and does notpresent a toxicity problem.

The biophysical characteristics of the HB_(s) Ag are well documented [J.Clinical Investigation 52, 1176 (1973), J. of Virology 10, 469 (1972)]as a negatively charged particle. In the presence of a very highconcentration of positively charged sodium ions there is formed asodium-HB_(s) Ag salt molecule. This type of molecule is compatible withthe human biological system. In contrast to prior art products, theHB_(s) Ag of the preferred mode of carrying out the present invention issubstantially free of other cations, particularly added cesium andpotassium ions.

The superior solubility of NaBr at lowered temperatures with respect toKBr permits the use of lowered temperatures more conductive to stabilityof biological materials. The use of a step gradient rather than a lineargradient is preferred as it accumulates impurities at the stepboundaries and permits processing a larger volume of plasma in a singlegradient.

The antigen of the present invention is useful per se as an antigen forhepatitis B and can be used as described in U.S. Pat. No. 3,636,191. TheHB_(s) Ag antigen of the the present invention is a highly purifiedproduct which has been shown to be substantially free of blood groupsubstances A and B as measured by serological and electrophoresistechniques. In addition, the antigen of the present invention can beused as the starting material for the hepatitis B antigen of copendingapplication Ser. No. 577,483, filed May 14, 1975, now U.S. Pat. No.4,017,360. Where the starting material is plasma the isopycnic bandingstep results in about a 100 fold purification of HB_(s) Ag relative tonormal plasma protein, and the rate zonal step results in about afurther 20 fold purification of HB_(s) Ag relative to normal plasmaprotein. The combination of the two steps result in about a 2000 foldpurification of HB_(s) Ag relative to normal plasma protein.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

The rotor of a centrifuge, Electronucleonics K, is filled with 8,400 mlof phosphate buffer. After running the rotor up to 10,000 rpm to degasthe system, the following step gradient is pumped into the bottom of thestationary rotor:

1. 2,400 ml of 10% NaBr, ρ=1.08

2. 1,000 ml of 20% NaBr, ρ=1.17

3. 1,500 ml of 30% NaBr, ρ=1.28

4. 3,500 ml of 40% NaBr, ρ=1.41

Plasma containing Australia antigen (HB_(s) Ag), 1,750 ml, is pumpedinto the top of the stationary rotor displacing 1,750 ml of 40% NaBrfrom the bottom of the rotor. The rotor is accelerated to 30,000 rpm andrun at this speed for 18 hours. After stopping the rotor 500 ml ofHB_(s) Ag rich material in the 1.21-1.24 density region, is collectedand dialyzed against phosphate buffer.

The rotor is then filled with phosphate buffer, degassed as above, andthe following step gradient pumped into the bottom of the stationaryrotor:

1. 2,400 ml of 5% sucrose, ρ=1.02

2. 1,750 ml of 15% sucrose, ρ=1.06

3. 1,750 ml of 25% sucrose, ρ=1.10

4. 2,500 ml of 50% sucrose, ρ=1.23

The HB_(s) Ag rich material from the NaBr isopycnic banding step, 500ml, is pumped into the rotor top displacing 500 ml. of 50% sucrose outthe rotor bottom. The rotor is then run at 28,000 rpm for 18 hours.After stopping the rotor, 500 ml of HB_(s) Ag rich material in the1.135-1.165 density region is collected.

EXAMPLE 2

The rotor of a centrifuge, Electronucleonics K, is filled with 8,400 mlof phosphate buffer. After running the rotor up to 10,000 rpm to degasthe system, the following step gradient is pumped into the bottom of thestationary rotor:

1. 2,400 ml of 10% NaBr, ρ=1.08

2. 1,000 ml of 20% NaBr, ρ=1.17

3. 1,500 ml of 30% NaBr, ρ=1.28

4. 3,500 ml of 40% NaBr, ρ=1.41

Plasma containing HB_(s) Ag, 1,750 ml, is pumped into the top of thestationary rotor displacing 1,750 ml of 40% NaBr from the bottom of therotor. The rotor is accelerated to 30,000 rpm and run at this speed for4 hours. The rotor is then stopped and 1,750 ml of 40% NaBr are pumpedinto the bottom of the rotor forcing the plasma out the top. Anadditional 1,750 ml of fresh plasma containing HB_(s) Ag are pumped intothe top of the rotor displacing an equal volume of 40% NaBr out thebottom of the rotor. The rotor is then run at 30,000 rpm for 18 hours.After stopping the rotor 1,000 ml of HB_(s) Ag rich material in the1.21-1.24 density region is collected and dialyzed against phosphatebuffer.

The rotor is then filled with phosphate buffer, degassed as above, andthe following step gradient pumped into the bottom of the stationaryrotor:

1. 2,400 ml of 5% sucrose, ρ=1.02

2. 1,750 ml of 15% sucrose, ρ=1.06

3. 1,750 ml of 25% sucrose, ρ=1.10

4. 2,500 ml of 50% sucrose, ρ=1.23

The HB_(s) Ag rich material from the NaBr isopycnic banding step, 1,000ml, is pumped into the rotor top displacing 1,000 ml. of 50% sucrose outthe rotor bottom. The rotor is then run at 28,000 rpm for 18 hours.After stopping the rotor, 1,000 ml of HB_(s) Ag rich material in the1.135-1.165 density region is collected.

EXAMPLE 3

The rotor of a centrifuge, Electronucleonics K, is filled with 8,400 mlof phosphate buffer. After running the rotor up to 10,000 rpm to degasthe system, the following step gradient is pumped into the bottom of thestationary rotor:

1. 2,400 ml of 10% NaBr, ρ=1.08

2. 1,000 ml of 20% NaBr, ρ=1.17

3. 1,500 ml of 30% NaBr, ρ=1.28

4. 3,500 ml of 40% NaBr, ρ=1.41

Plasma containing HB_(s) Ag, 1,750 ml, is pumped into the top of thestationary rotor displacing 1,750 ml of 40% NaBr from the bottom of therotor. The rotor is accelerated to 30,000 rpm and run at this speed for4 hours. The rotor is then stopped and 1,750 ml of 40% NaBr are pumpedinto the bottom of the rotor forcing the plasma out the top. Anadditional 1,750 ml of fresh plasma containing HB_(s) Ag are pumped intothe top of the rotor displacing an equal volume of 40% NaBr out thebottom of the rotor. The rotor is accelerated to 30,000 rpm and run atthis speed for 4 hours. The rotor is then stopped and a third charge of1,750 ml of fresh plasma containing HB_(s) Ag are pumped into the top ofthe rotor displacing an equal volume of 40% NaBr out the bottom of therotor. The rotor is then run at 30,000 rpm for 18 hours. After stoppingthe rotor, 1,500 ml of HB_(s) Ag rich material in the 1.21-1.24 densityregion is collected and dialyzed against phosphate buffer.

The rotor is then filled with phosphate buffer, degassed as above, andthe following step gradient pumped into the bottom of the stationaryrotor:

1. 2,400 ml of 5% sucrose, ρ=1.02

2. 1,750 ml of 15% sucrose, ρ=1.06

3. 1,750 ml of 25% sucrose, ρ=1.10

4. 2,500 ml of 50% sucrose, ρ=1.23

The HB_(s) Ag rich material from the NaBr isopycnic banding step, 1,500ml, is pumped into the rotor top displacing 1,500 ml of 50% sucrose outthe rotor bottom. The rotor is then run at 28,000 rpm for 18 hours.After stopping the rotor 1,500 ml of HB_(s) Ag rich material in the1.135 1.165 density region is collected.

EXAMPLE 4

The following table shows the marked increase in yield per unit of timewhen using the multiple loading technique of the present invention(Examples 2 and 3) as compared with single loading (Example 1).

    ______________________________________                                                       Total isopycnic                                                                           % Increase                                                                             % Increase                                       Yield   and rate zonal                                                                            in time (with                                                                          in yield with                                    (ml) of centrifuging                                                                              respect to                                                                             respect to                                Example                                                                              HB.sub.s Ag                                                                           time (hours)                                                                              Example 1                                                                              Example 1)                                ______________________________________                                        1       500    36          --       --                                        2      1,000   40          11.1%    100%                                      3      1,500   44          22.2%    200%                                      ______________________________________                                    

EXAMPLE 5

The final product of Example 2 prepared under aseptic conditions tocontain 30-40 μg of HB_(s) Ag measured by Lowry protein method istreated with 1:4000 formaldehyde at 37° C. for 72 hours. Excess residualformaldehyde is neutralized with sodium bisulfite. All treatments areperformed under aseptic conditions. The product is stored at 5° C.Intramuscular injection in guinea pigs of three doses of 1 ml given a 1month intervals induces circulatory hepatitis B surface antibody.

EXAMPLE 6

The product of Example 5 after inactivation is adsorbed on alum andsuspended in physiological saline solution buffered at from pH 6.0 toabout pH 7.8. This product is stored at 5° C. Intramuscular injectionsin guinea pigs of three doses of 1 ml given at 1 month intervals inducescirculatory hepatitis B surface antibodies.

EXAMPLE 7

The product of Example 5 is administered to a group of individualspositive for hepatitis B surface antibody by PHA assay to induce a hightiter (2,000 PHA units/ml or greater) of hepatitis B surface antibody.The antibody titer of these individuals ranged from 20-1,000 PHAunits/ml. The individuals are administered 1 ml doses of the product ofExample 5 intramuscularly. Initial immunication schedule is 0 days, 1month, 3 months. Booster injections are given at 4 month intervals. Thevaccinees are monitored by PHA assay and those displaying a significantincrease of HB_(s) Ab (2,000 PHA units/ml or greater) are selected asplasma donors. The resulting plasma is pooled and used to prepare immuneglobulin (HBIG) having a known titer of hepatitis B surface antibody.

EXAMPLE 8

The product of Example 6 is administered to a group of individualspositive for hepatitis B surface antibody by PHA assay to induce a hightiter (2,000 PHA units/ml or greater) of hepatitis B surface antibody.The antibody titer of these individuals ranged from 20-1,000 PHAunits/ml. The individuals are administered 1 ml doses of the product ofExample 6 intramuscularly. Initial immunization schedule is 0 days, 1month, 3 months. Booster injections are given at 4 month intervals. Thevaccinees are monitored by PHA assay and those displaying a significantincrease of HB_(s) Ab (2,000 PHA units/ml or greater) are selected asplasma donors. The resulting plasma is pooled and used to prepare immuneglobulin (HBIG) having a known titer of hepatitis B surface antibody.

What is claimed is:
 1. A method of preparing hepatitis B immune globulinwhich comprisesselecting a plurality of individuals positive forhepatitis B surface antibody, administering to the individuals hepatitisB surface antigen in an amount effective to stimulate antibodyformation, monitoring the antibody level of the individuals followingadministration of the hepatitis B surface antigen, obtaining plasma fromthose individuals exhibiting an increase of hepatitis B surface antibodyof at least 2,000 PHA units/ml, and preparing hepatitis B immuneglobulin from the plasma.